Fig. 4

Soluble CD146 binds integrin αvβ3 on U87 cells. The expression of angiomotin (Amot) and αvβ3 on U87 cells was analyzed by flow cytometry (a). The effect of silencing RNA targeting αv, β3, αL and β2 was analyzed on sCD146-induced U87 cell proliferation (b). U87 cells were transfected with siRNA targeting αv, β3, αL and β2 and sCD146-FITC binding was determined by flow cytometry (c). CHO cells were co-transfected with plasmids encoding integrin subunits αv and β3 or control plasmids (mock) and sCD146-FITC binding was determined by flow cytometry (d) or by immunofluorescence microscopy (e). Mock transfected or αvβ3 expressing CHO cells were treated or not with blocking anti-αvβ3 antibody and then sCD146 was added for 1 h at 37°. CD146 was examined after immunoprecipitation with anti- αvβ3 antibody. Loading was analyzed using IgG heavy chain (f). In two ELISA assays, the effect of cyclic RGD peptide/mucizumab (g) and the dissociation constant Kd (h) were estimated for the binding of αvβ3 to rsCD146. White bars correspond to 25 µm. Average of 3 experiments is shown; *p < 0.05, **p < 0.01, ***p < 0.001, experimental vs control